Characterization of the Binding Performance of Silica-Based Immunoaffinity Adsorbents Prepared via Online Coupling Reactions

Abstract

Antibodies against a conjugate of daidzein and bovine serum albumin (BSA) were immobilized onto dihydrazide-activated silica beads via online coupling reactions. The binding performances of the obtained immunoaffinity adsorbents to the corresponding hapten and carrier protein were all optimized and characterized. It was found that online coupling reactions offered a convenient and practical way to generate the requisite immunoaffinity columns. The optimum binding buffers for BSA and daidzein were found to be pH 5.0, 0.01 mol L−1 phosphate-buffered saline (PBS) and 0.05% Tween-20 in pure water, indicating different interactions between the immobilized antibodies with different parts of the antigen. Under the optimum operating conditions, the silica-based immunoaffinity adsorbents showed specific and selective binding properties to the target protein; while for small molecules, a mixed adsorption mechanism caused by both specific and non-specific interactions was observed. For comparison, offline coupled silica-antibody immunoaffinity adsorbents and Sepharose 4B support immobilized with the same antibody were also prepared and tested. The experimental results of this study may provide useful information for further development of other high-performance immunoaffinity chromatographic methods for different purposes.